PHYTOCHEMICAL ANALYSIS AND BIOLOGICAL ACTIVITY STUDIES OF AN EASTERN CAPE MEDICINAL PLANT, STRYCHNOS HENNINGSII BY MONDELI MNGOMA DISSERTATION SUBMITTED IN FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE MAGISTER TECHNOLOGIAE IN CHEMISTRY IN THE FACULTY OF SCIENCE AT THE NELSON MANDELA UNIVERSITY SUPERVISOR: DR B. G. HLANGOTHI DECLARATION I, Mondeli Mngoma (student number: 211106682), hereby declare that the dissertation for Magister Technologiae (MTech) is my own work and that it has not previously been submitted for assessment or completion of any postgraduate qualification to another University or for another qualification. Mondeli Mngoma ACKNOWLEDGEMENTS I would like to thank the following people; My supervisor, Dr B. Hlangothi for all her hard work, assistance and motivation. This work would not only be impossible without a supervisor like you, and I would not have discovered my love for the laboratory. I sincerely thank you. I thank my mother every day, for everything she’s ever done for our family. She is and will always be our rock. I also thank my siblings, who made TTUMMS as strong as it is today. My whole family for cheering me on when I would be caught between a rock and a hard place. My friends who have helped me try and enjoy this little thing called life. The BGH research family, who have helped me gain confidence and laugh at my jokes. The Botany, Biochemistry departments and Dr R. Betz’s group for all their assistance. The Nelson Mandela Metropolitan University for their assistance and opportunities they have presented my way. The National Research Foundation (NRF) for financially supporting aspiring researchers all over South Africa. ABSTRACT This project sees the chemical investigation of an Eastern Cape medicinal plant, Strychnos henningsii. The aim of this project was to investigate the phytoconstituents and activity of organic extracts of S. henningsii as well as isolation and characterization of single compounds. S. henningsii is one of the most widely used tree bark in the Eastern Cape in treating a variety of ailments. Evaluation of the traditional herbal use of the S. henningsii bark was warranted. Both the ethyl acetate and methanol extract proved to possess non-toxic properties and showed cell growth potential at low concentrations. The anti- inflammatory response of both extracts showed appreciable results, and they did not promote inflammatory due to the alkaloidal presence. Although the extracts both showed the presence of phenols, the anti-oxidant capacity by DPPH, FRAP, and ORAC assays was considerately lower than expected. The ethyl acetate extract presented a compound thought to be a terpenoid (4.1). Three compounds were isolated from the methanol extract; two alkaloidal compounds and one acrylate. The alkaloids were isolated from the methanol extract, stryvomicine (4.2) and an isovomicine derivative (4.3). Their structures were deduced from NMR (1D and 2D experiments) and HRMS spectra. This acrylate was found to be 3-(4- methylphenyl)acrylic acid via single-crystal XRD, and this is the first report of it being isolated from the Strychnos genus. Profiling of S. henningsii was performed via Liquid Chromatography–Mass Spectroscopy. Other spectroscopic techniques utilised in the interpretation of isolated compounds included Nuclear Magnetic Resonance, Infrared, single-crystal X-Ray Diffraction and High Resolution Mass spectroscopy. Table of Contents Chapter One: Introduction .................................................................................... 1 1.1 Natural product background ................................................................................. 1 1.1.1 Phytochemicals ............................................................................................. 2 1.2 Purpose of the Study ............................................................................................ 3 1.3 Botanical description of Strychnos henningsii ...................................................... 4 Plant parts used ............................................................................................. 5 Traditional use ............................................................................................... 5 1.4 Why the Strychnos henningsii of the Eastern Cape Province? ............................ 6 1.5 Chapters Overview ............................................................................................... 6 1.6 References ........................................................................................................... 7 Chapter Two: Literature Review ........................................................................ 10 2.1 Introduction ........................................................................................................ 10 2.2 Traditional herbal medicine in Africa .................................................................. 10 2.3 Traditional herbal medicine in South Africa ........................................................ 11 2.4 The Eastern Cape region ................................................................................... 13 2.5 The Loganiaceae family ..................................................................................... 15 2.6 Strychnos species .............................................................................................. 18 2.6.1 Known compounds from Strychnos species ................................................ 18 2.7 Strychnos henningsii .......................................................................................... 23 2.7.1 Chemical investigations of Strychnos henningsii ......................................... 23 2.7.2 Biological assays ......................................................................................... 26 2.8 References ......................................................................................................... 27 Chapter Three: Experimental ............................................................................. 31 3.1 Plant material ..................................................................................................... 31 3.2 Preparation of bark extracts ............................................................................... 31 3.3 Chromatography ................................................................................................ 32 3.3.1 Analytical thin-layer chromatography ........................................................... 32 3.3.2 Liquid chromatography ................................................................................ 32 3.3.3 Column chromatography ............................................................................. 32 3.4 Phytochemical analysis ...................................................................................... 33 3.5 In vitro assays .................................................................................................... 35 3.5.1 Cytotoxicity .................................................................................................. 35 3.5.2 Anti-inflammatory ......................................................................................... 36 3.5.3 Anti-oxidant .................................................................................................. 36 3.5.3.1 DPPH radical scavenging assay ........................................................... 36 3.5.3.2 FRAP (Ferric Reducing Ability of Plasma) ............................................. 37 3.5.3.3 ORAC (Oxygen Radical Absorbance Capacity) .................................... 37 3.6 Analytic instruments ........................................................................................... 38 3.6.1 Nuclear Magnetic Resonance (NMR) .......................................................... 38 3.6.2 Liquid Chromatography–Mass Spectroscopy (LC-MS) ................................ 40 3.6.3 High-Resolution Mass Spectroscopy (HRMS) ............................................. 40 3.6.4 X-Ray Diffraction (XRD) ............................................................................... 40 3.6.5 Infrared (IR) ................................................................................................. 40 Chapter Four: Results and Discussion ............................................................. 42 4.1 Phytochemical analysis ...................................................................................... 42 4.2 Screening of crude extracts ................................................................................ 44 4.2.1 Cytotoxicity screening .................................................................................. 45 4.2.2 Anti-inflammatory screening ........................................................................ 47 4.2.3 Anti-oxidant assays ...................................................................................... 49 4.2.3.1 2,2-diphenyl-1-picrylhydrazyl (DPPH) ................................................... 50 4.2.3.2 FRAP (Ferric Reducing Ability of Plasma) ............................................. 52 4.2.3.3 ORAC (Oxygen Radical Absorbance Capacity) .................................... 54 4.3 Spectroscopic analysis of isolated compounds .................................................. 57 4.3.1 Analysis of isolated compounds .................................................................. 57 4.3.1.1 Stryvomicine (4.2) ................................................................................. 63 4.3.1.2 Isovomicine (4.3) ..................................................................................